The proposed research concerns the molecular mechanisms which determine and regulate the transport of sodium and potassium across the membranes of normal, mature human red blood cells. The primary focus of the studies is to determine the effect of the voltage across the cell membrane on the linear and saturable components of passive cation transport as well as on active transport. Because of their relative structural and functional simplicity, human red blood cells are an advantageous tissue for relating voltage to fluxes of electrolytes. In the proposed study, a variety of chemical voltage clamp techniques will be tested and developed. A computer model of red cell ionic equilibria will be used to aid in defining conditions and controls which ensure that the membrane potential is the only significant variable. For passive transport, unidirectional tracer influxes and effluxes, as well as net fluxes, will be compared with predictions of the flux ratio equation and the constant field theory. In this manner, the dependence on voltage of ionic permeabilities which describe the ground fluxes, as well as the affinities and maximal velocities which describe the mediated passive and active fluxes will be determined. A variety of types of cation fluxes, as defined by pharmacological reagents and other conditions, will be examined. In addition, the effects of temperature and ionic strength on passive fluxes will be considered. Model systems, including resealed ghost membranes, inside-out vesicles, and native and reconstituted "Band III" vesicles, will also be employed in an attempt to simplify some of the technique and interpretations. The essential rationale for the proposed investigation is that filling the gap in our knowledge of the voltage dependence of passive and active cation transport in normal, mature human red blood cells will further our understanding of transport mechanisms; the new information and techniques will then be available for future studies of altered patterns of transport which are known to exist in developing and pathological forms of red blood cells.